A new and unexpected domain-domain interaction in the AraC protein

An interaction between the dimerization domains and DNA binding domains of the dimeric AraC protein has previously been shown to facilitate repression of the Escherichia coli araBAD operon by AraC in the absence of arabinose. A new interaction between the domains of AraC in the presence of arabinose is reported here, the regulatory consequences of which are unknown. Evidence for the interaction is the following: the dissociation rate of arabinose-bound AraC from half-site DNA is considerably faster than that of free DNA binding domain, and the affinity of the dimerization domains for arabinose is increased when half-site DNA is bound. In addition, an increase in the fluorescence intensity of tryptophan residues located in the arabinose-bound dimerization domain is observed upon binding of half-site DNA to the DNA binding domains. Direct physical evidence of the new domain-domain interaction is demonstrated by chemical crosslinking and NMR experiments.     

Inhibition of TATA binding protein dimerization by RNA polymerase III transcription initiation factor Brf1

The Brf1 subunit of TFIIIB plays an important role in recruiting the TATA-binding protein (TBP) to the up-stream region of genes transcribed by RNA polymerase III. When TBP is not bound to promoters, it sequesters its DNA binding domain through dimerization. Promoter assembly factors therefore might be required to dissociate TBP into productively binding monomers. Here we show that Saccharomyces cerevisiae Brf1 induces TBP dimers to dissociate. The high affinity TBP binding domain of Brf1 is not sufficient to promote TBP dimer dissociation but in addition requires the TFIIB homology domain of Brf1. A model is proposed to explain how two distinct functional domains of Brf1 work in concert to dissociate TBP into monomers.     

Influence of the N-terminal domain and divalent cations on self-association and DNA binding by the Saccharomyces cerevisiae TATA binding protein

The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K+, and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the full-length protein.     

Structure and properties of a truely apo form of AraC dimerization domain

The arabinose-binding pockets of wild type AraC dimerization domains crystallized in the absence of arabinose are occupied with the side chains of Y31 from neighboring domains. This interaction leads to aggregation at high solution concentrations and prevents determination of the structure of truely apo AraC. In this work we found that the aggregation does not significantly occur at physiological concentrations of AraC. We also found that the Y31V mutation eliminates the self-association, but does not affect regulation properties of the protein. At the same time, the mutation allows crystallization of the dimerization domain of the protein with only solvent in the arabinose-binding pocket. Using a distance difference method suitable for detecting and displaying even minor structural variation among large groups of similar structures, we find that there is no significant structural change in the core of monomers of the AraC dimerization domain resulting from arabinose, fucose, or tyrosine occupancy of the ligand-binding pocket. A slight change is observed in the relative orientation of monomers in the dimeric form of the domain upon the binding of arabinose but its significance cannot yet be assessed.     

The C-terminal end of AraC tightly binds to the rest of its domain

Genes were synthesized to express two DNA binding domains of AraC connected by short linkers. The abilities of the resulting proteins to bind to DNA containing AraC half-sites separated by the usual four bases as well as an additional two or three helical turns of the DNA were measured. The inability of some of the protein constructs to bind to widely separated half-sites indicates that the C-terminal 14 amino acids of AraC are firmly bound to the rest of the DNA binding domain.     

UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.     

Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N‐terminal domain

Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites – the N‐terminal domain (Site‐I) and the extracellular/transmembrane domain (Site‐II). Therefore, higher monomer affinity could be due to stronger binding at Site‐I or Site‐II or both. We have now characterized the binding of a human CXCR1 N‐terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N‐domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with ～10‐ to 100‐fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site‐I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function.     

Recombinant human retinoic acid receptor alpha. Binding of DNA and synthetic retinoids to the protein expressed in Escherichia coli.

The human retinoic acid receptor alpha was expressed in Escherichia coli. The recombinant protein was found to be very unstable in several E. coli strains, probably due to proteolysis. Conditions were established to obtain reasonable amounts of active protein for ligand and DNA binding studies. The recombinant receptor showed the expected DNA binding activities in gel-retardation assays. Ligand binding properties were measured in a charcoal absorption assay. The dissociation constant for highly specific bound retinoic acid was found to be 0.67 nM. The affinity of several synthetic retinoids to the recombinant protein was determined and compared to their biological activity. Some of the values presented here differ significantly from those published earlier for the receptor or its isolated hormone-binding domain.     

Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain.

In this study, we have investigated the influence of regions outside the DNA-binding domain of the human glucocorticoid receptor on high-affinity DNA binding. We find that the DNA-binding domain shows a 10-fold lower affinity for a palindromic DNA-binding site than the intact receptor. The N-terminal part of the receptor protein does not influence its DNA-binding affinity, while the C-terminal steroid-binding domain increases the DNA-binding affinity of the receptor molecule. It has previously been shown that both the intact glucocorticoid receptor and the glucocorticoid receptor DNA-binding domain bind to a palindromic glucocorticoid response element on DNA as dimers. It is likely that differences in DNA-binding affinity observed result from protein-protein interactions outside the DNA-binding domain between receptor monomers, as has been shown for the estrogen receptor. We have previously identified a segment involved in protein-protein interactions between DNA-binding domains of glucocorticoid receptors. This, in combination with results presented in this study, suggests that there are at least two sites of contact between receptor monomers bound to DNA. We suggest that the interaction between the DNA-binding domains may act primarily to restrict DNA binding to binding sites with appropriate half-site spacing and that additional stability of the receptor dimer is provided by the interactions between the steroid-binding domains.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.

The possibility of increasing the affinity of a Taq DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A. A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq DNA polymerase in the range of 30-50 nM as determined by biosensor technology. Analysis of binding data indicated that the increases in affinity were predominantly due to decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Taq DNA polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Taq DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.     

Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns

We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.     

Multimerization of the Drosophila zeste protein is required for efficient DNA binding.

The Drosophila zeste protein forms multimeric species in vitro through its C-terminal domain. Multimerization is required for efficient binding to DNA containing multiple recognition sequences and increasing the number of binding sites stimulates binding in a cooperative manner. Mutants that can only form dimers still bind to a dimeric site, but with lower affinity. Mutations or progressive deletions from the C-terminal show that when even dimer formation is prevented, DNA-binding activity is lost. Surprisingly, binding activity is regained with larger deletions that leave only the DNA-binding domain. Additional protein sequences apparently inhibit DNA binding unless they permit multimerization. The DNA-binding domain peptides bind strongly even to isolated recognition sequences and they bind as monomers. The ability of various zeste peptides to stimulate white gene expression in vivo shows that multimeric forms are the functional species of the zeste product in vivo. The DNA-binding domain peptide binds well to DNA in vitro, but it cannot stimulate white gene expression in vivo. This failure may reflect the need for an activation domain or it may be caused by indiscriminate binding of this peptide to non-functional isolated sites. Multimerization increases binding specificity, selecting only sites with multiple recognition sequences.     

Identification of soluble interleukin-1 binding protein in cell-free supernatants. Evidence for soluble interleukin-1 receptor

This study describes the identification and characterization of a soluble interleukin-1 (IL-1) binding protein in the conditioned media from Raji human B-lymphoma cells. The soluble IL-1 binding material was isolated by IL-1 affinity chromatography, and treatment with trypsin decreased its ability to bind to IL-1 demonstrating its protein nature. The soluble IL-1 binding protein was specific for IL-1 and was able to discriminate between Il-1 alpha and IL-1 beta in a manner analogous to the membrane-bound Raji IL-1 receptor. The specificity of the IL-1 binding protein was further established in two ways. 1) Cell-free supernatants from Raji "receptor-negative" cells did not contain any IL-1 binding protein, thus ruling out nonspecific interactions between IL-1 and a serum or other protein present in the conditioned medium; and 2) the soluble binding protein inhibited IL-1 binding to Raji cells in a dose-dependent manner. Scatchard analysis of IL-1 beta binding showed the dissociation constant (KD) to be 5.1 nM for the soluble IL-1 binding protein compared with 0.8 nM for the membrane-bound IL-1 receptor. Gel chromatography of the soluble binding protein yielded a major peak of IL-1 binding activity with a molecular mass of 35-45 kDa. The characteristics of the soluble IL-1 binding protein described above are consistent with those of the extracellular binding domain of the membrane-bound Raji IL-1 receptor.     

DNA binding affinity and sequence permutation preference of the telomere protein from Euplotes crassus

Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single-strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins, we have characterized the DNA binding properties of the telomere end binding protein from Euplotes crassus (EcTEBP). EcTEBP and its predicted amino-terminal DNA-binding domain, EcTEBP-N, were expressed in Escherichia coli and purified. Each protein formed stoichiometric (1:1) complexes with single-strand DNA oligos derived from the precisely defined d(TTTTGGGGTTTTGG) sequence found at DNA termini in Euplotes. Dissociation constants for DNA x EcTEBP and DNA x EcTEBP-N complexes were comparable: K(D-DNA) = 38 +/- 2 nM for the full-length protein and K(D-DNA) = 60 +/- 4 nM for the N-terminal domain, indicating that the N-terminal domain retains a high affinity for DNA even in the absence of potentially stabilizing moieties located in the C-terminal domain. Rate constants for DNA association and DNA dissociation corroborated a slightly improved DNA binding performance for the full-length protein (ka = 45 +/- 4 microM(-1) s(-1), kd = 0.10 +/- 0.02 s(-1)) relative to that of the N-terminal domain (ka = 18 +/- 1 microM(-1) s(-1), kd = 0.15 +/- 0.01 s(-1)). Equilibrium dissociation constants measured for sequence permutations of the telomere repeat spanned the range of 55-1400 nM, with EcTEBP and EcTEBP-N binding most tightly to d(TTGGGGTTTTGG), the sequence corresponding to that of mature DNA termini. Additionally, competition experiments showed that EcTEBP recognizes and binds the telomere-derived 14-nucleotide DNA in preference to shorter 5'-truncation variants. Compared with the results for multisubunit complexes assembled with telomere single-strand DNA from Oxytricha nova, our results highlight the relative simplicity of the E. crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single-strand DNA.